ARTICLE Received 16 Apr 2013 | Accepted 8 Aug 2013 | Published 16 Sep 2013 DOI: 10.1038/ncomms3420 OPEN Genome signature-based dissection of human gut metagenomes to extract subliminal viral sequences Lesley A. Ogilvie1, Lucas D. Bowler1, Jonathan Caplin2, Cinzia Dedi1, David Diston2,w, Elizabeth Cheek3, Huw Taylor2, James E. Ebdon2 & Brian V. Jones1 Bacterial viruses (bacteriophages) have a key role in shaping the development and functional outputs of host microbiomes. Although metagenomic approaches have greatly expanded our understanding of the prokaryotic virosphere, additional tools are required for the phage- oriented dissection of metagenomic data sets, and host-range affiliation of recovered sequences. Here we demonstrate the application of a genome signature-based approach to interrogate conventional whole-community metagenomes and access subliminal, phylogen- etically targeted, phage sequences present within. We describe a portion of the biological dark matter extant in the human gut virome, and bring to light a population of potentially gut- specific Bacteroidales-like phage, poorly represented in existing virus like particle-derived viral metagenomes. These predominantly temperate phage were shown to encode functions of direct relevance to human health in the form of antibiotic resistance genes, and provided evidence for the existence of putative ‘viral-enterotypes’ among this fraction of the human gut virome. 1 Centre for Biomedical and Health Science Research, School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton BN2 4GJ, UK. 2 School of Environment and Technology, University of Brighton, Brighton BN2 4GJ, UK. 3 School of Computing, Engineering and Mathematics, University of Brighton, Brighton BN2 4GJ, UK. w Present address: Mikrobiologische and Biotechnologische Risiken Bundesamt fu¨r Gesundheit BAG, 3003 Bern, Switzerland. Correspondence and requests for materials should be addressed to B.V.J. (email: [email protected]). NATURE COMMUNICATIONS | 4:2420 | DOI: 10.1038/ncomms3420 | www.nature.com/naturecommunications 1 & 2013 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3420 iruses are the most abundant infectious agents on the phage–host species or closely related organisms. The latter should planet, and collectively constitute a highly diverse and facilitate inference of host-range and permit a more in-depth Vlargely unexplored gene-space, which accounts for much analysis of the local ecological landscape populated by recovered of the ‘biological dark matter’ in Earth’s biosphere1–3. Bacterial phage, and together with the former stands to provide an viruses (bacteriophage or phage) are considered the most alternative and novel perspective on the gut virome. Therefore, numerous viral entities, and through their effects on host whole-community metagenomes may constitute valuable bacteria, phage can influence processes ranging from global resources for the analysis of phage communities, and in geochemical cycles to bacterial virulence and pathogenesis1–5. conjunction with VLP-derived data sets, provide a more complete The study of this expansive family of viruses continues to understanding of phage concurrent with the human gut and other underpin many fundamental insights into microbial physiology ecosystems8. and evolution, with the interplay of bacteria and phage now Nevertheless, the resolution and host-range affiliation of phage studied at scales ranging from the individual components fragments present in conventional metagenomes remains challen- of single-phage species, to community-level surveys of viral ging, with particular problems arising from the paucity of well- assemblages and their impacts on host microbial ecosystems. characterized phage reference genomes with established host The development of metagenomic tools for analysis of phage ranges, a lack of universally conserved and robust phylogenetic populations constitutes a major advance in this regard, which is anchors in phage genomes (akin to bacterial 16S rRNA genes), as poised to deliver unprecedented insight into the prokaryotic well as the mosaic nature of phage genomes, and the fragmentary virosphere. This powerful culture-independent approach over- nature of metagenomic data sets8,13. These factors, in conjunction comes many limitations of traditional methods for phage with the potential value of standard metagenomes for virome isolation and characterization, ultimately promising almost analysis, highlight the need to develop robust approaches for unrestricted access to the genetic content of host microbiomes phage-oriented dissection of these repositories, and host-range and their attendant viral collectives3,6–11. Application of these affiliation of recovered phage sequences. techniques to the study of microbial viromes has already provided Here we demonstrate the application of a genome signature- major insights into a number of phage communities, including based approach for retrieval of subliminal, phylogenetically those associated with microbial ecosystems that develop in or on targeted phage sequences present within conventional gut the human body7,11,12. microbial metagenomes. Application of this strategy permitted In particular, the retinue of phage associated with the human the identification of a subset of gut-specific Bacteroidales-like gut microbiome is now increasingly recognized as an important phage sequences poorly represented in existing VLP-derived viral facet of this ecosystem, which may significantly influence its metagenomes. These phage sequences were shown to encode impact on human health3,5,13–16. Gut-associated phage have functions of direct relevance to human health, and provided new already been shown to encode genes that confer production of insights into the structure and composition of the human gut toxins, virulence factors or antibiotic resistance upon host virome. bacteria5,17,18, and have the potential to modulate community structure and metabolic output through elimination of host species or introduction of new traits1,16,19. Furthermore, virome Results composition also appears to be altered in disease states, which has Genome signature-based recovery of ‘Bacteroidales-like’ phage. given rise to the hypothesis that the human gut virome may Members of the Bacteroidales, and in particular the genus Bac- have a role in the pathogenesis of disorders associated with teroides, are abundant and important constituents of the human perturbation of the gut ecosystem14. Phage also hold considerable gut microbiome for which few complete phage genomes are biotechnological and pharmaceutical potential, with the gut available, with this region of the gut virome believed to remain virome now a viable target for bio-prospecting and the largely uncharted13. To more fully explore this novel phage gene- development of novel therapeutic or diagnostic tools3,13. space, we utilized Bacteroidales phage sequences as ‘drivers’ to However, current strategies for generating viral metagenomes interrogate 139 human gut metagenomes based on tetranucleotide are not without limitations, and are typically based on analysis usage profiles (TUPs) and functional profiles of contigs (Table 1, of nucleic acids derived from purified virus like particles Supplementary Figs S1–S3, Supplementary Table S1). (VLPs)3,7,11,20. As such, these approaches are targeted towards This strategy takes advantage of similarities in global nucleotide analysis of free-phage particles present at the time of sampling, usage patterns, or the genome signature, arising between phage which restricts access to the quiescent virome fraction and infecting the same or related host bacterial species22–24.Weexploit obscures host-range information8. VLP-based approaches will this phenomenon to identify contigs related to Bacteroidales phage also poorly represent phage not efficiently recovered during driver sequences in assembled gut metagenomes, and subsequent virion purification stages, and typically rely on subsequent function-based binning to resolve phage fragments recovered in amplification of extracted viral DNA before sequencing, which this process (Fig. 1). We refer to this strategy as phage genome can also exclude some phage types3,7,11,20. Although these caveats signature-based recovery (PGSR), and denote sequences obtained do not undermine the overall utility of the VLP approach (which in this way with the PGSR prefix. retains a clear advantage in accessing actively replicating phage), Interrogation of all large contigs (10 kb and over) from human much scope remains to develop complementary strategies to gut metagenomes (Supplementary Table S1) recovered 408 access and analyse microbial viromes. metagenomic fragments with TUPs similar to Bacteroidales In this context, it is notable that conventional metagenomic phage drivers. Eighty five fragments were categorized as phage data sets, derived from total community DNA, have been found based on functional profiling, and the remainder classified as to contain significant fractions of phage sequence data, and in the non-phage (presumed chromosomal, n ¼ 320), or could not be case of the gut microbiome, this has been estimated to be up to categorized (n ¼ 3) (Supplementary Data 1). The proportion of 17% of microbial DNA recovered from stool samples7,11,21. sequences categorized as phage within the total pool of 408 Owing to the focus on acquisition of chromosomal sequences and sequences recovered by PGSR (20.83%; 85/408) is congruent with an independence